Rotor gene q mdx 5plex hrm instrument

High-risk HPV testing was done using the HPV-Risk assay (Self-screen B.V., Amsterdam), which is a multiplex, real-time PCR assay targeting the E7 region of 15 (probably) HR-HPV types (HPV16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -66, -67, and -68), and providing additional genotype information for HPV16 and HPV18 [42 (link)]. The assay includes amplification of the reference gene HBB to assess DNA quality and quantity. The assay was performed using 5 µL of the DNA extract according to the manufacturer’s instructions, on a Rotor-Gene Q MDx 5plex HRM instrument (Qiagen GMBH, Hilden, Germany). The Cq value cut-offs of the assay for calling a sample HPV-positive were not used. Samples were scored HPV positive when there was a Cq value for any of the HPV targets.

In brief, 5 μL of genomic DNA was added to 20 μL of the amplification mix according to manufacturer’s instructions (Cat. No.: 674023, QIAGEN GmbH, Hilden, Germany) using the Universal Taqman PCR Master Mix (Applied Biosystem). PCR reaction was programmed as follow: 50 °C for 2 min, 95 °C for 10 min followed by 45 cycles of 95 °C for 15 s (denaturation) and 60 °C for 1 min (Annealing/extension) followed by High-resolution melting (HRM) using Rotor-Gene^® Q MDx 5plex HRM instrument (QIAGEN GmbH, Hilden, Germany).

FAM19A4/miR124‐2 methylation analysis including sample DNA extraction was performed as previously described.²⁴ For bisulfite‐conversion, the EZ DNA Methylation Kit was used according to the manufacturer's specifications (Zymo Research, Irvine, California).³² Bisulfite‐converted DNA was subsequently used as input for quantitative PCR analysis of the FAM19A4 and miR124‐2 promoter methylation levels using the QIAsure Methylation Test (QIAGEN). For all centers, a sample input of 2.5 μL bisulfite‐converted DNA was used for PCR on the Rotor‐Gene Q MDx 5plex HRM instrument (QIAGEN) equipped with the AssayManager software (QIAGEN). This software runs the assay followed by automatic quality assurance and data analysis. The reported results were hypermethylation‐positive, negative or invalid. Additional quality assurance was employed using the housekeeping gene β‐actin (ACTB) as a reference for successful bisulfite‐conversion, sample quality and signal normalization. Methylation testing was performed by local technicians blinded for the clinical data.